Clostridium perfringens is an ubiquitous bacterium that can colonise a variety of different biotopes. Due to its anaerobic lifestyle it is not surprising to find C. perfringens as a commensal of the normal gut flora in humans and domesticated animals. However, under certain circumstances it is known to be responsible for causing some severe diseases due to its production of a wide range of toxins (Songer (1996) Clin Microbiol Rev vol 9: 216-234). Apart from the four toxins used for typing C. perfringens (alpha-, beta-, epsilon-, iota-toxin) it is able to produce a selection of non-typing toxins, such as enterotoxin or perfringolysin O (Petit et al., (1999) Trends Microbiol vol 7: 104-110). Recently, a novel toxin named NetB (Necrotic enteritis toxin B) has been identified and suggested to play a role in the pathogenesis of avian necrotic enteritis (NE), a severe gastro-intestinal disease that manifests in gross lesions within the intestines of poultry (WO2008/148166). NE is a re-emerging disease that is causing enormous economic costs to the worldwide poultry industry (around 2 billion US dollars per year) (Keyburn et al., (2008) PLoS Pathog vol 4: e26). Its re-emergence is due to the initiative of some governments to prohibit the use of antimicrobial growth promoters in animal feed, amongst others to reduce the evolving spread of antibiotic-resistant bacteria in the environment.
The NetB is produced by C. perfringens toxinotype A strains and, to a lesser extent, by strains of type C (Kaldhusdal et al. (1999) FEMS Immunol Med Microbiol vol 24: 337-343). The protein is 322 amino acids long in its active form and has an estimated molecular weight of 36.5 kDa. Although the molecular basis of toxicity is still little understood, several studies suggest that the NetB is a new member of the small β-pore-forming toxins (β-PFTs) as it is able to form pores on membranes and shares amino acid sequence similarity with several other related members of the small pore-forming toxins family (38% identity with the beta toxin from C. perfringens, 40% identity with the C. perfringens delta toxin, and 31% identity with the alpha toxin from S. aureus) (Keyburn et al. (2008) PLoS Pathog vol 4: e26; Manich et al. (2008) PLoS One vol 3: e3764). It was initially assumed that the alpha toxin, which is produced by the same bacterium, is the major virulence factor for causing NE, but experiments with an alpha toxin mutant showed that this strain was still virulent and able to cause disease (Keyburn et al. (2006) Infect Immun vol 74: 6496-6500). In contrast, a netB mutant was not capable of causing NE, whereas the wild type and the complemented mutant could (Keyburn et al. (2008) PLoS Pathog vol 4: e26). However, it is still unsettled as to whether the NetB is the key virulence factor for causing NE, as in some cases it was reported that even C. perfringens strains without the netB gene were still capable of virulence (Cooper & Songer (2009) Vet Microbiol vol 142: 323-328). Moreover, immunization studies with alpha toxin and other antigens, such as a hypothetical zinc metalloprotease and a pyruvate-ferredoxine oxidoreductase, have been identified to moderately protect chicken from developing NE (Cooper et al. (2009) Vet Microbiol vol 133: 92-97; Zekarias et al. (2008) Clin Vaccine Immunol vol 15: 805-816; Kulkarni et al. (2010) Clin Vaccine Immunol vol 17: 205-214; Kulkarni et al. (2007) Clin Vaccine Immunol vol 14: 1070-1077).
The heptameric structure of one of the most widely studied β-PFT, S. aureus α-hemolysin (αHL), was determined over 20 years ago (Song L et al. (1996) Science vol. 274: 1859-1866) and was, until recently, the only high resolution structure of a β-PFT in the membrane-inserted form. The ring-shaped complex resembles a mushroom with the cap forming the extracellular domain and the stem forming the membrane-spanning region, in which each subunit contributes one β-hairpin. Although NetB appears to form pores in target cell membranes, little is known about the molecular basis for this toxicity which hinders the development of effective control measures against NE.
Several attempts have been made on the development of an effective vaccine to protect chicken against NE but, to date, without significant success.